Name: isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation
Uploaded: 2015-04-29T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

この作品は、自然科学とカナダの工学研究評議会（NSERC、何の298515から2011を付与していない）からの助成金によってサポートされていました。 ALは博士の受信者であります自然科学とカナダの工学研究評議会（NSERC）から奨学金、およびMSは、カナダ衛生研究所からの助成金からサポートされていました（無許可、CIHR。MOP123246）。

すべての手順は、センターデRECHERCHEデュセンターHospitalierドゥモントリオール大学（CRCHUM）の動物実験委員会の承認を得た後、動物飼育ガイドラインにカナダ議会に従って行われました。 1.単離、同定、およびマウス腹腔マクロファージの文化<ol><li> 3.8％のビールチオグリコール酸培地を準備します。これを行うには、蒸留水1000mlにチオグリコール酸培地の38グラムを中断。完全培地を溶解するために沸騰するソリューションを持参してください。 121℃で15分間オートクレーブで滅菌します。 RT 18で、暗所で3ヶ月まで保管してください。 注：濁度は細菌汚染を示している開発した場合は廃棄してください。無菌に保つ場合に溶液は暗所で1年まで維持することができます。 </li><li>各畝の腹腔内に3.8％ブリューワーチオグリコール酸培地の1ミリリットルを注入し、23 Gの針に取り付けられた1ミリリットル注射器を使用して、SEと3日間待ちます。各マウスについて、新しい注射器と針を使用してください。 </li><li>ペントバルビタールナトリウム（100mg / kg）の腹腔内注射によりマウスを麻酔し、頸椎脱臼によりマウスを安楽死させます。呼吸数をチェックして、適切な麻酔を確認してください。通常、急速な呼吸マウスが深く麻酔されていないことを示しています。 </li><li> 70％エタノールで各マウスの腹部を洗います。はさみを使用して、腹膜の下正中線に沿って横方向の切開を行います。 </li><li>鉗子を使用して、透明な腹膜肌を露出させるために腹部の皮膚を引き戻します。 20 G針に取り付けられた5ミリリットルの注射器を用いて、各マウスの腹腔内に冷DPBSを5ml注入します。 </li><li>腹膜腔に穏やかなマッサージを行い、その後、任意の器官を穿刺することなく、慎重に流体を吸引します。針を外し、50mlのコニカル遠心チューブに腹水を分注します。 </li><li>冷却遠心機で400×gで10分間遠心分離します。細胞は、全手順の間に冷たいままにする必要があります。上清を捨て、RPMI培地1640中で細胞ペレットを再懸濁します。 </li><li>血球計数器を使用して、細胞をカウントし、1×10 6細胞/ mlの細胞密度に調整します。 </li><li> F4 / 80に対するマウスとantibodieあたり1×10 6個の細胞（マクロファージに発現される表面抗原）を用いてフローサイトメトリーにより単離された細胞の表現型を特徴づけます。 </li></ol> 2.細胞処置<ol><li>直接、単離後、各ウェルに1×10 6個の細胞を追加します。 37℃で1〜2時間のためにそれらを培養することにより、6ウェルプレートに付着したマウス腹腔マクロファージのままにしておきます。優しく温かいPBSで3回洗浄して非接着細胞を除去します。 </li><li>次のTLRリガンドの存在下で、24時間900μlの無血清DMEM中で続いて、培養細胞：（Pam3CSK4-TLR1 / 2（0.5 mg / mlの）;ポリ（I：C）-TLR3（10 mg / mlの） ; LPS-TLR4（100ng / mlの）フラジェリンTLR5（100ng / mlの）FSL1-TLR6 / 2（100ng / mlの） ssRNA40-TLR7（1 / mlの）; ODN1826-TLR9（1μM）。 注：各リガンドのための10×ストック溶液を準備します。このように10倍希釈を行うことにより、各ウェルに、後者の100μlを添加します。 </li></ol> 3. R​​NAの単離<ol><li>各ウェルに1ミリリットルのTRIzolを添加し、ピペットを介して細胞溶解液を数回通過させることにより、6ウェルプレート中で直接すべての培地および溶解細胞を除去します。核タンパク質複合体を完全に解離可能にするために、室温で5から10分間均質化したサンプルをインキュベートします。 </li><li> 1.5ミリリットルのRNaseおよびDNaseをフリーのマイクロ遠心チューブにライセートを転送します。 1ミリリットルののTRIzolあたり0.2ミリリットルのクロロホルムを追加します。 15秒間手で激しく管を振って5から10分間、室温でそれらを培養します。 4℃で15分間、12,000×gで遠心分離します。下の赤相（フェノール - クロロホルム）、間期、無色上部の水相に分離する混合物を観察します。 RNAは水相でのみ残ります。 </li><li>トラン相間を乱すことなく、新鮮なチューブにsferは慎重に上部の水相。イソプロピルアルコール0.5 mlを混合して、そこからRNAを沈殿させます。 4℃で10分間、12,000×gで10分間遠心分離室温でサンプルをインキュベートします。 RNAは、チューブの側面と底に白い沈殿物を形成する沈殿します。 </li><li>上清を取り除きます。 75％エタノール1mlで一度RNAペレットを洗浄します。 4℃で5分間、7,500×gでボルテックスし、遠心分離することにより、サンプルを混ぜます。簡単に言えば、RNA（10分間空気乾燥）を乾燥させます。 0.1ミリリットルDEPC（RNアーゼを含まない水）でRNAを溶解し、55℃で10分間インキュベートします。 </li><li>分光光度計を用いてRNAを定量化します。これを行うには、サンプルをDEPC水で100倍に希釈し、260nmの波長で読み取りました。 RNA濃度は、その後、次のようになります。OD 260×40 NG / UL X希釈係数を。 </li><li> RNA濃度を均一化し、第一鎖cDNA合成キットをRT-PCRシステムを用いて、製造業者の指示に従ってcDNAを合成。として製造者の指示に従って、リアルタイムDNA検出システムでリアルタイムPCR（45サイクル）によって遺伝子のmRNAレベルを測定します。例えばβアクチンおよびGAPDHのための2つのハウスキーピング遺伝子との遺伝子発現レベルを標準化。 注：ミリリットルを500μg/にRNA濃度を正規化し、cDNA合成のための0.5μgを使用します。 </li></ol>

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The authors have no competing financial interests.

マクロファージは、生存のために重要であり、免疫学的目的のためのホストを操作するための魅力的な標的を提供します。のTLRおよび他の認識分子の発見は、免疫学的議論の中心にマクロファージを行いました。マクロファージは、サイトカイン、ダメージ関連分子パターン分子（DAMPS）20および病原体（のPAMPs）21のグループに関連する分子を含む種々の刺激に応答します。?...

細網内皮系の貪食は、骨髄、血液、肝臓および脾臓のような種々の組織および器官中の細胞から構成されています。マクロファージは広く、彼らは特に制御する自然免疫と適応免疫応答と明確な感染症に関与体の周りに分布しています。宿主防御におけるそれらの役割に加えて、マクロファージはまた、創傷治癒および組織の恒常性1,2-維持に重要な役割を果たしています。さらに、マクロファージだけでなく、免疫機能に重要であるだけでなく、積極的に鉄ホメオスタシス3に参加しています。本体では、鉄の約80％は、老化がマクロファージ4によって貪食される赤血球内のヘモグロビンに存在します。毎日、これらのマクロファージは赤血球由来の鉄25mgをリサイクルし、プラズマ5にその輸送を提供しています。また、感染および炎症の間、前炎症性マクロファージ鉄availabiliを減少させるために血清鉄を封鎖します両方の全身および地域レベル6-8で病原体へのTY。マクロファージおよび主に肝細胞が鉄代謝9、10のマスターレギュレーターであると考えられるヘプシジンという名前の抗菌ペプチドを生成することだけでなく、研究が示されています。ヘプシジンは、主に炎症性刺激によって増加し、慢性炎症11-13時のマクロファージにおける鉄の隔離のために部分的に責任があるされています。マクロファージにおけるヘプシジンの発現は非常によく理解されていないとして、我々はこの調節におけるToll様受容体（TLR）の役割の可能性を検討しました。 TLRは主にマクロファージで発見し、それらの活性化において中心的な役割を果たしています。また、肝臓におけるLPS誘導性のヘプシジン発現は、TLR4 13に依存しています。したがって、我々の研究を実行するために、我々は、マウスの腹腔マクロファージの単離に基づく方法を使用していました。 マクロファージ細胞株は広くマクロファージスタッドに使用されていますIES;それにもかかわらず、拡張培養は、遺伝子の損失を誘発し、これらの細胞株における免疫機能を低下させることができます。このように、腹腔からマクロファージの単離は非常に重要です。 マウス腹腔マクロファージ13-15を収穫するのに理想的なサイトを紹介します。単離されたマウス腹腔マクロファージは、その免疫学的機能に関するいくつかの研究のために便利です。しかし、腹膜におけるマクロファージの数は、鋭意研 ​​究のために不十分であり、マウス当たり約1×10 6のマクロファージが推定されます。したがって、マクロファージの出力を上げるために、例えば、チオグリコール酸のような滅菌誘発剤は、細胞の収穫の前に腹腔内に注射しました。チオグリコレート注射後、マウスのマクロファージあたりの収量は10倍増加しました。マクロファージ収量、マクロファージの動員、マサチューセッツ州で、その結果、炎症反応を誘導する刺激としてビールチオグリコール酸培地行為の増加にもかかわらず、Yが、必須ではないが、遺伝子発現に影響を​​与えます。したがって、未処理マクロファージからなる対照群を各実験に含めなければなりません。私たちの手で、非常に炎症によって刺激されるヘプシジン発現は、非処置チオグリコール酸では検出されなかった腹腔マクロファージを誘発しました。また、研究では、ビールチオグリコール酸は、多数のマクロファージを動員するが、それらに16を活性化しないことが示されています。一方、ブリューワーのチオグリコール酸は、マクロファージは、リソソーム酵素の増加が、摂取した微生物17を殺すの減少を示した誘発しました。非誘発マクロファージ16と比較した場合しかし、貪食能は影響を受けませんでした。 皿で培養した後、腹腔マクロファージは、したがって、腹膜腔から単離された細胞の他のタイプからそれらの分離を可能にする、接着性になります。続いて、単離されたマクロファージは、異なるTLRのアゴニストで攻撃しました。最後に、mRNAを培養細胞から抽出し、遺伝子発現は、定量的逆転写酵素ポリメラーゼ連鎖反応（QRT-PCR）を用いて分析しました。

<table><tbody><tr><td>C57BL/6 mice</td><td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td><td>475</td><td></td></tr><tr><td>Thioglycollate</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>19032-500G</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td><td>WISENT INC Canada (QC)</td><td>311-425-CL</td><td></td></tr><tr><td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td><td>WISENT INC Canada (QC)</td><td>350-000-CL</td><td></td></tr><tr><td>1 and 5 ml syringes</td><td>BD USA (NJ)</td><td>309659</td><td></td></tr><tr><td>6-well plates</td><td>Corning Incorporated (NY, USA)</td><td>MCT-150-C</td><td></td></tr><tr><td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-pm25</td><td></td></tr><tr><td>Polyionosine&amp;ndash;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-PIC</td><td></td></tr><tr><td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>L2880</td><td></td></tr><tr><td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FLIC-10</td><td></td></tr><tr><td>Lipoprotein synthetic FSL1 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FSL</td><td></td></tr><tr><td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &amp;mu;g/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-LRNA-40</td><td></td></tr><tr><td>Type B CpG oligonucleotide ODN1826 (1 &amp;mu;M)</td><td>InvivoGen (San Diego, USA)</td><td>11B16-MM</td><td></td></tr><tr><td>TRIzol</td><td>Invitrogen, (Burlington, ON, Canada)</td><td>15596-026</td><td></td></tr><tr><td>20 G and 23 G needles</td><td>BD USA (NJ)</td><td>305175</td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tubes placed on ice</td><td>Sarstedt (Newton, MA, USA)</td><td>62.547.205</td><td></td></tr><tr><td>Red Blood Cells Lysis Buffer</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>R7757-100ML</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>F4/80 antibody</td><td>BIO-RAD ( CA, USA)</td><td>MCA497APC</td><td></td></tr><tr><td>CD16/CD32 antibodies</td><td>Pharmingen, {Mississauga, ON, CA)</td><td>553141</td><td></td></tr><tr><td>Flow cytometer</td><td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Axygen Scietific (CA,USA)</td><td>3516</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific (ON, Canada)</td><td>UN1888</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>JT Baker (PA, USA)</td><td>70566</td><td></td></tr><tr><td>75% ethanol (in DEPC treated water)</td><td>Commercial Alchohols (QC, Canada)</td><td>17394</td><td></td></tr><tr><td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>216.542.8</td><td></td></tr><tr><td>Omniscript RT-PCR system</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>205113</td><td></td></tr><tr><td>Rotor Gene 3000</td><td>Montreal Biotech, (Kirkland, QC, Canada)</td><td></td><td></td></tr><tr><td>QuantiTect SYBR Green I PCR kits</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>204141</td><td></td></tr></tbody></table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

感染症や炎症の間、循環単球は、血流を離れ、彼らはマクロファージに分化する組織へと移行します。マクロファージは、微生物の広い範囲の進化を通じて保存された分子パターンを認識し、表面のToll様受容体（TLR）を発現します。 TLRは、通常、遺伝子発現の変化に関連しているマクロファージの活性化において中心的な役割を果たしています。マクロファージは、多くの疾患において重要であり、治療のための魅力的な標的として浮上しています。以下のプロトコルでは、我々はビールチオグリコール酸培地を用いてマウスの腹腔マクロファージを単離するための手順が記載されています。後者は、それに応じて、これは10倍マクロファージ収量を発生させます、腹膜への単球の移行を後押しします。いくつかの研究は、骨髄、脾臓または腹腔由来マクロファージを用いて行われてきました。しかし、腹腔マクロファージは、分離の際に、より成熟したことが示されており、それらの機能に、より安定しているし、性と表現型。このように、マウスの腹腔から単離されたマクロファージは、異なる免疫学的および代謝の研究に役立つことができる重要な細胞集団を提示します。単離されると、マクロファージは、異なるTLRリガンドで刺激し、その結果、遺伝子発現を評価しました。

まず、フローサイトメトリーによって単離されたマウス腹腔マクロファージを特徴とします。これを行うには、我々は、具体的にのみマクロファージによって発現されるマーカーを認識する（F4 / 80）抗体を使用していました。この特徴付けは、単離されたマクロファージの割合を決定し、分離プロセス中に得られた細胞の間で区別するために必要とされます。 （ 図1）に示され?...

Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Toll様受容体刺激時の遺伝子発現解析を行うためにマウス腹腔マクロファージの単離

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

isolation-murine-peritoneal-macrophages-to-carry-out-gene-expression

Research

JoVE Journal

Immunology and Infection

36.3K ビュー.  Institut du cancer de Montréal. We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

ビデオ: Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

マウス腹腔細胞の分離

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

免疫・感染学

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

成人肝細胞の混合初代培養からマクロファージを分離するためにシンプルかつ効率的な方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

透過性膜支持体との共培養を用いたマクロファージ活性化に及ぼす腫瘍分泌パラクリンリガンドの効果の研究

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

がん研究

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

ダブル勾配遠心、テフロンコーティングされた細胞培養バッグ中のマクロファージへの分化によるヒト単球の単離

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

マウス骨髄と腹腔マクロファージ活性化抗体を用いた薬剤効果の評価

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

トール様受容体シグナル伝達の阻害剤に対する食作用性免疫細胞における生物活性ナノ粒子のスクリーニング

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

医学

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

マクロファージに貪食アポトーシス胸腺細胞の実験的解析

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

マウス由来骨髄由来マクロファージの単離と培養

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Source: Zhen, Y. et al., <a href="https://app.jove.com/v/59731">Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

腹膜マクロファージによるアポトーシス性胸腺細胞のエフェロサイトーシスの解析

Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure ...